Glucagon analogues

ABSTRACT

The invention provides materials and methods for the treatment of obesity and excess weight, diabetes, and other associated metabolic disorders. In particular, the invention provides novel glucagon analog peptides effective in such methods. The peptides may mediate their effect by having increased selectivity for the GLP-1 receptor as compared to human glucagon.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/701,952, filed on Sep. 17, 2012, European Application No. 12184744.6,filed on Sep. 17, 2012, and U.S. Provisional Application No. 61/784,294,filed on Mar. 14, 2013, which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to glucagon analogues and their medicaluse, for example in the treatment of obesity and excess weight,diabetes, and other metabolic disorders.

BACKGROUND OF THE INVENTION

Pre-proglucagon is a 158 amino acid precursor polypeptide that isdifferentially processed in the tissues to form a number of structurallyrelated proglucagon-derived peptides, including glucagon (Glu),glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), andoxyntomodulin (OXM). These molecules are involved in a wide variety ofphysiological functions, including glucose homeostasis, insulinsecretion, gastric emptying and intestinal growth, as well as regulationof food intake.

Glucagon is a 29-amino acid peptide that corresponds to amino acids 53to 81 of pre-proglucagon. Oxyntomodulin (OXM) is a 37 amino acid peptidewhich includes the complete 29 amino acid sequence of glucagon with anoctapeptide carboxyterminal extension (amino acids 82 to 89 ofpre-proglucagon, and termed “intervening peptide 1” or IP-1. The majorbiologically active fragment of GLP-1 is produced as a 30-amino acid,C-terminally amidated peptide that corresponds to amino acids 98 to 127of pre-proglucagon.

Glucagon helps maintain the level of glucose in the blood by binding toglucagon receptors on hepatocytes, causing the liver to releaseglucose—stored in the form of glycogen—through glycogenolysis. As thesestores become depleted, glucagon stimulates the liver to synthesizeadditional glucose by gluconeogenesis. This glucose is released into thebloodstream, preventing the development of hypoglycemia.

GLP-1 decreases elevated blood glucose levels by improvingglucose-stimulated insulin secretion and promotes weight loss chieflythrough decreasing food intake.

OXM is released into the blood in response to food ingestion and inproportion to meal calorie content. OXM has been shown to suppressappetite and inhibit food intake in humans (Cohen et al, Journal ofEndocrinology and Metabolism, 88, 4696-4701, 2003; WO 2003/022304). Inaddition to those anorectic effects, which are similar to those ofGLP-1, OXM must also affect body weight by another mechanism, since ratstreated with oxyntomodulin show less body weight gain than pair-fed rats(Bloom, Endocrinology 2004, 145, 2687). Treatment of obese rodents withOXM also improves their glucose tolerance (Parlevliet et al, Am JPhysiol Endocrinol Metab, 294, E142-7, 2008) and suppresses body weightgain (WO 2003/022304).

OXM activates both the glucagon and the GLP-1 receptors with a two-foldhigher potency for the glucagon receptor over the GLP-1 receptor, but isless potent than native glucagon and GLP-1 on their respectivereceptors. Human glucagon is also capable of activating both receptors,though with a strong preference for the glucagon receptor over the GLP-1receptor. GLP-1 on the other hand is not capable of activating glucagonreceptors. The mechanism of action of oxyntomodulin is not wellunderstood. In particular, it is not known whether some of theextrahepatic effects of the hormone are mediated through the GLP-1 andglucagon receptors, or through one or more unidentified receptors.

Other peptides have been shown to bind and activate both the glucagonand the GLP-1 receptor (Hjort et al, Journal of Biological Chemistry,269, 30121-30124, 1994) and to suppress body weight gain and reduce foodintake (see, for example, WO 2006/134340, WO 2007/100535, WO 2008/10101,WO 2008/152403, WO 2009/155257, WO 2009/155258, WO2010/070252,WO2010/070253, WO2010/070255, WO2010/070251, WO2011/006497,WO2011/160630, WO2011/160633).

Obesity is a globally increasing health problem is associated withvarious diseases, particularly cardiovascular disease (CVD), type 2diabetes, obstructive sleep apnea, certain types of cancer, andosteoarthritis. As a result, obesity has been found to reduce lifeexpectancy. According to 2005 projections by the World HealthOrganization there are 400 million adults (age>15) classified as obeseworldwide. In the US, obesity is now believed to be the second-leadingcause of preventable death after smoking.

The rise in obesity drives an increase in diabetes, and approximately90% of people with type 2 diabetes may be classified as obese. There are246 million people worldwide with diabetes, and by 2025 it is estimatedthat 380 million will have diabetes. Many have additional cardiovascularrisk factors, including high/aberrant LDL and triglycerides and low HDL.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a compound having the formula:R¹—X—Z—R²

wherein

R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R² is OH or NH₂;

X is a peptide which has the formula:

(SEQ ID NO: 1) H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLS (SEQ ID NO: 2)HSQGTFTSDYSKYLDSKAAHDFVEWLLSA (SEQ ID NO: 4)H-Aib-QGTFTSDYSKYLDEKAAKDFIEWLLSA (SEQ ID NO: 5)H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLESA (SEQ ID NO: 6)HSQGTFTSDYSRYLDSKAAEDFVEWLLRA (SEQ ID NO: 7)HSQGTFTSDYSKYLDSKAAEDFVEWLLRA (SEQ ID NO: 8)HSQGTFTSDYSKYLDSKAAHDFVEWLLS (SEQ ID NO: 9) HSQGTFTSDYSKYLDSKAAHDFVEWLLR(SEQ ID NO: 10) HSQGTFTSDYSKYLDEKAAHEFVEWLESA (SEQ ID NO: 11)H-Aib-QGTFTSDYSKYLDEKRAKDFIEWLLS (SEQ ID NO: 12)HSQGTFTSDYSRYLDSKAAHDFVEWLLSA (SEQ ID NO: 17)H-Aib-HGTFTSDYSKYLESKAAEEFIEWLESA (SEQ ID NO: 18)HSHGTFTSDYSKYLEEKAAHEFIEWLESA (SEQ ID NO: 19)H-Aib-HGTFTSDYSKYLEEKAAHEFVEWLESA

and Z is absent or is a sequence of 1-20 amino acid units independentlyselected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu,Lys, Arg, Dbu, Dpr and Orn; or a pharmaceutically acceptable salt orsolvate thereof.

In a second aspect, the invention provides a compound of the formula:R¹—X—OH

wherein

R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

and X is a peptide of SEQ ID NO: 3, 13 or 14:

(SEQ ID NO: 3) HSQGTFTSDYSKYLDSKAAHDFVEWLLRA (SEQ ID NO: 13)H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLSA (SEQ ID NO: 14)H-Aib-QGTFTSDYSKYLDEKRAKDFIEWLLSA

or a pharmaceutically acceptable salt or solvate thereof.

In a third aspect, the invention provides a compound having the formula:R¹—X—Z—R²

wherein

R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R² is OH or NH₂;

X is a peptide of SEQ ID NO: 15:

(SEQ ID NO: 15) H-Aib-QGTFTSDYSKYLDEKAAKDFIEWLESA

or which differs from SEQ ID NO: 15 at up to three of the followingpositions whereby, if different from SEQ ID NO: 15:

the residue at position 2 is selected from Ser and D-Ser

the residue at position 3 is selected from His and Hse

the residue at position 12 is Arg

the residue at position 15 is Glu

the residue at position 16 is Ser

the residue at position 18 is Arg

the residue at position 20 is selected from His and Glu

the residue at position 21 is Glu

the residue at position 23 is Val

the residue at position 28 is selected from Arg and Glu

the residue at position 29 is absent;

and Z is absent or is a sequence of 1-20 amino acid units independentlyselected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu,Lys, Arg, Dbu, Dpr and Orn;

or a pharmaceutically acceptable salt or solvate thereof.

In certain embodiments, the residue at position 20 is not variable andis His.

The compound may have the formula:R¹—X—Z—R²

wherein

R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R² is OH or NH₂;

X is a peptide of SEQ ID NO: 15:

(SEQ ID NO: 15) H-Aib-QGTFTSDYSKYLDEKAAKDFIEWLESA

or which differs from SEQ ID NO: 15 at one or both of the followingpositions whereby, if different from SEQ ID NO: 15:

the residue at position 21 is Glu

the residue at position 23 is Val

and Z is absent or is a sequence of 1-20 amino acid units independentlyselected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu,Lys, Arg, Dbu, Dpr and Orn;

or a pharmaceutically acceptable salt or solvate thereof.

In a fourth aspect, the invention provides a compound having theformula:R¹—X—Z—R²

wherein

R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R² is OH or NH₂;

X is a peptide of SEQ ID NO: 16:

(SEQ ID NO: 16) H-Aib-QGTFTSDYSKYLDSKAAEDFVEWLESA

or which differs from SEQ ID NO: 16 at up to three of the followingpositions whereby, if different from SEQ ID NO: 16:

the residue at position 2 is selected from Ser and D-Ser

the residue at position 3 is selected from His and Hse

the residue at position 12 is Arg

the residue at position 15 is Glu

the residue at position 16 is Glu

the residue at position 18 is Arg

the residue at position 20 is selected from Lys and His

the residue at position 21 is Glu

the residue at position 23 is Ile

the residue at position 28 is selected from Arg and Glu

the residue at position 29 is absent

and Z is absent or is a sequence of 1-20 amino acid units independentlyselected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu,Lys, Arg, Dbu, Dpr and Orn; or a pharmaceutically acceptable salt orsolvate thereof.

The compound may have the formula:R¹—X—Z—R²

wherein

R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R² is OH or NH₂;

X is a peptide of SEQ ID NO: 16:

(SEQ ID NO: 16) H-Aib-QGTFTSDYSKYLDSKAAEDFVEWLESA

or which differs from SEQ ID NO: 16 at one or both of the followingpositions whereby, if different from SEQ ID NO: 16:

the residue at position 21 is Glu

the residue at position 23 is Ile

and Z is absent or is a sequence of 1-20 amino acid units independentlyselected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu,Lys, Arg, Dbu, Dpr and Orn; or a pharmaceutically acceptable salt orsolvate thereof.

A compound of the third or fourth aspects of the invention may comprisea peptide X having the sequence:

(SEQ ID NO: 15) H-Aib-QGTFTSDYSKYLDEKAAKDFIEWLESA (SEQ ID NO: 16)H-Aib-QGTFTSDYSKYLDSKAAEDFVEWLESA (SEQ ID NO: 20)H-Aib-QGTFTSDYSKYLEEKAAKDFIEWLESA (SEQ ID NO: 21)H-Aib-QGTFTSDYSKYLESKAAEDFIEWLESA (SEQ ID NO: 22)HSQGTFTSDYSKYLEEKAAKDFIEWLESA (SEQ ID NO: 23)H-Aib-QGTFTSDYSKYLESKAAHDFVEWLESA  (SEQ ID NO: 24)H-Aib-QGTFTSDYSKYLESKAAEDFVEWLESA or (SEQ ID NO: 25)H-DSer-QGTFTSDYSKYLDEKAAKDFIEWLESA.

An any aspect, the compound of the invention may have the formula:

H-H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLS-OH H-HSQGTFTSDYSKYLDSKAAHDFVEWLLSA-OHH-H-Aib-QGTFTSDYSKYLDEKAAKDFIEWLLSA-NH₂H-H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLESA-NH₂H-HSQGTFTSDYSRYLDSKAAEDFVEWLLRA-NH₂ H-HSQGTFTSDYSKYLDSKAAEDFVEWLLRA-NH₂H-HSQGTFTSDYSKYLDSKAAHDFVEWLLS-NH₂ H-HSQGTFTSDYSKYLDSKAAHDFVEWLLR-OHH-HSQGTFTSDYSKYLDEKAAHEFVEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLDEKRAKDFIEWLLS-OHH-HSQGTFTSDYSRYLDSKAAHDFVEWLLSA-NH₂ H-HSQGTFTSDYSKYLDSKAAHDFVEWLLRA-OHH-H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLSA-OHH-H-Aib-QGTFTSDYSKYLDEKRAKDFIEWLLSA-OHH-H-Aib-QGTFTSDYSKYLDEKAAKDFIEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLDSKAAEDFVEWLESA-NH₂H-Aib-HGTFTSDYSKYLESKAAEEFIEWLESA-OH H-HSHGTFTSDYSKYLEEKAAHEFIEWLESA-OHH-H-Aib-HGTFTSDYSKYLEEKAAHEFVEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLEEKAAKDFIEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLESKAAEDFIEWLESA-NH₂H-HSQGTFTSDYSKYLEEKAAKDFIEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLESKAAHDFVEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLESKAAEDFVEWLESA-NH₂H-H-DSer-QGTFTSDYSKYLDEKAAKDFIEWLESA-NH₂

or may be a pharmaceutically acceptable salt or solvate thereof.

For the avoidance of doubt, in compounds of the third and fourth aspectsof the invention, those positions which are not expressly stated topermit variability are intended to be fixed and thus only include thestated residue at those positions.

In all aspects of the invention, one or more of the amino acid sidechains in the peptide X or Z (where present) may be conjugated to alipophilic substituent.

Preferably, one or more of the amino acid side chains in the peptide Xis conjugated to the lipophilic substituent.

A lipophilic substituent may have the formula Z¹ wherein Z¹ is alipophilic moiety conjugated (covalently linked) directly to the sidechain of the relevant residue of X or Z, or Z¹Z² where Z¹ is alipophilic moiety, Z² is a spacer, and Z¹ is conjugated to the sidechain of the residue of X or Z via Z².

Additionally or alternatively, one or more of the amino acid side chainsin peptide X or Z (where present) is conjugated to a polymericsubstituent.

In certain embodiments, the peptide X or X—Z carries only one lipophilicsubstituent and/or only one polymeric substituent.

Lipophilic and polymeric substituents are described in more detailbelow.

The lipophilic moiety and/or polymeric substituent may be conjugated tothe side chain of any suitable residue. A lysine residue may beparticularly suitable, e.g. a lysine residue at position 12 (if present)or 17.

Thus peptide X may have the formula:

H-Aib-QGTFTSDYSKYLDSK*AAHDFVEWLLS HSQGTFTSDYSKYLDSK*AAHDFVEWLLSAH-Aib-QGTFTSDYSKYLDEK*AAKDFIEWLLSA H-Aib-QGTFTSDYSKYLDSK*AAHDFVEWLESAHSQGTFTSDYSRYLDSK*AAEDFVEWLLRA HSQGTFTSDYSKYLDSK*AAEDFVEWLLRAHSQGTFTSDYSKYLDSK*AAHDFVEWLLS HSQGTFTSDYSKYLDSK*AAHDFVEWLLRHSQGTFTSDYSKYLDEK*AAHEFVEWLESA H-Aib-QGTFTSDYSKYLDEK*RAKDFIEWLLSHSQGTFTSDYSRYLDSK*AAHDFVEWLLSA HSQGTFTSDYSKYLDSK*AAHDFVEWLLRAH-Aib-QGTFTSDYSKYLDSK*AAHDFVEWLLSA H-Aib-QGTFTSDYSKYLDEK*RAKDFIEWLLSAH-Aib-QGTFTSDYSKYLDEK*AAKDFIEWLESA H-Aib-QGTFTSDYSKYLDSK*AAEDFVEWLESAH-Aib-HGTFTSDYSKYLESK*AAEEFIEWLESA HSHGTFTSDYSKYLEEK*AAHEFIEWLESAH-Aib-HGTFTSDYSKYLEEK*AAHEFVEWLESA H-Aib-QGTFTSDYSKYLEEK*AAKDFIEWLESAH-Aib-QGTFTSDYSKYLESK*AAEDFIEWLESA HSQGTFTSDYSKYLEEK*AAKDFIEWLESAH-Aib-QGTFTSDYSKYLESK*AAHDFVEWLESA H-Aib-QGTFTSDYSKYLESK*AAEDFVEWLESAH-DSer-QGTFTSDYSKYLDEK*AAKDFIEWLESA

where “*” indicates the position of a lipohilic or polymericsubstituent, particularly a lipophilic substituent.

The compound of the invention may have the formula:

H-H-Aib-QGTFTSDYSKYLDSK*AAHDFVEWLLS-OHH-HSQGTFTSDYSKYLDSK*AAHDFVEWLLSA-OHH-H-Aib-QGTFTSDYSKYLDEK*AAKDFIEWLLSA-NH₂H-H-Aib-QGTFTSDYSKYLDSK*AAHDFVEWLESA-NH₂H-HSQGTFTSDYSRYLDSK*AAEDFVEWLLRA-NH₂H-HSQGTFTSDYSKYLDSK*AAEDFVEWLLRA-NH₂ H-HSQGTFTSDYSKYLDSK*AAHDFVEWLLS-NH₂H-HSQGTFTSDYSKYLDSK*AAHDFVEWLLR-OH H-HSQGTFTSDYSKYLDEK*AAHEFVEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLDEK*RAKDFIEWLLS-OHH-HSQGTFTSDYSRYLDSK*AAHDFVEWLLSA-NH₂ H-HSQGTFTSDYSKYLDSK*AAHDFVEWLLRA-OHH-H-Aib-QGTFTSDYSKYLDSK*AAHDFVEWLLSA-OHH-H-Aib-QGTFTSDYSKYLDEK*RAKDFIEWLLSA-OHH-H-Aib-QGTFTSDYSKYLDEK*AAKDFIEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLDSK*AAEDFVEWLESA-NH₂H-H-Aib-HGTFTSDYSKYLESK*AAEEFIEWLESA-OHH-HSHGTFTSDYSKYLEEK*AAHEFIEWLESA-OHH-H-Aib-HGTFTSDYSKYLEEK*AAHEFVEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLEEK*AAKDFIEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLESK*AAEDFIEWLESA-NH₂H-HSQGTFTSDYSKYLEEK*AAKDFIEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLESK*AAHDFVEWLESA-NH₂H-H-Aib-QGTFTSDYSKYLESK*AAEDFVEWLESA-NH₂H-H-DSer-QGTFTSDYSKYLDEK*AAKDFIEWLESA-NH₂

or may be a pharmaceutically acceptable salt or solvate thereof.

In certain embodiments, X has the formula:

H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLSHSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLSAH-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLLSAH-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLESAHSQGTFTSDYSRYLDS-K(Hexadecanoyl-isoGlu)- AAEDFVEWLLRAHSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAEDFVEWLLRAHSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLSHSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLRHSQGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAHEFVEWLESAH-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- RAKDFIEWLLSHSQGTFTSDYSRYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLSAHSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLRAH-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLSAH-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- RAKDFIEWLLSAH-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESAH-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAEDFVEWLESAH-Aib-HGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEEFIEWLESAHSHGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAHEFIEWLESAH-Aib-HGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAHEFVEWLESAH-Aib-QGTFTSDYSKYLEEK-(Hexadecanoyl-isoGlu)- AAKDFIEWLESAH-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEDFIEWLESAHSQGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESAH-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAHDFVEWLESAH-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEDFVEWLESAH-DSer-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA

The compound of the invention may be:

(Compound 1) H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLS-OH (Compound 2) H-HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-OH (Compound 4)H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLLSA-NH₂(Compound 5) H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLESA-NH₂ (Compound 6) H-HSQGTFTSDYSRYLDS-K(Hexadecanoyl-isoGlu)-AAEDFVEWLLRA-NH₂ (Compound 7) H-HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAEDFVEWLLRA-NH₂ (Compound 8) H-HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLS-NH₂ (Compound 9) H-HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLR-OH (Compound 10) H-HSQGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-AAHEFVEWLESA-NH₂ (Compound 11)H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- RAKDFIEWLLS-OH(Compound 12) H-HSQGTFTSDYSRYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ (Compound 3) H-HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLRA-OH (Compound 13)H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLSA-OH(Compound 14) H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-RAKDFIEWLLSA-OH (Compound 15)H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA-NH₂(Compound 16) H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAEDFVEWLESA-NH₂ (Compound 17)H-Aib-HGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEEFIEWLESA-OH(Compound 18) H-HSHGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAHEFIEWLESA-OH(Compound 19) H-H-Aib-HGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)-AAHEFVEWLESA-NH₂ (Compound 20)H-H-Aib-QGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA-NH₂(Compound 21) H-H-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)-AAEDFIEWLESA-NH₂ (Compound 22)H-HSQGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA-NH₂(Compound 23) H-H-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)-AAHDFVEWLESA-NH₂ (Compound 24)H-H-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEDFVEWLESA-NH₂(Compound 25) H-H-DSer-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-AAKDFIEWLESA-NH₂

or may be a pharmaceutically acceptable salt or solvate thereof.

For those peptide sequences X or X—Z composed exclusively ofnaturally-occurring amino acids, the invention further provides anucleic acid (which may be DNA or RNA) encoding a peptide X or X—Z asdefined herein. Also provided is an expression vector comprising such anucleic acid, and a host cell containing such a nucleic acid orexpression vector. The host cell is typically capable of expressing andoptionally secreting the encoded peptide X or X—Z.

The compounds of the invention are glucagon analogue peptides.References herein to a glucagon analogue peptide should be construed asreferences to a compound of the invention or to a peptide X or X—Z asthe context requires. Reference to a compound of the invention should betaken to include any pharmaceutically acceptable salt (e.g. an acetateor chloride salt) or solvate thereof, unless otherwise stated orexcluded by context.

The invention provides a composition comprising a compound of theinvention as defined herein (including pharmaceutically acceptable saltsor solvates thereof, as already described), a nucleic acid encoding apeptide X or X—Z, an expression vector comprising such a nucleic acid,or a host cell containing such a nucleic acid or expression vector, inadmixture with a carrier. In preferred embodiments, the composition is apharmaceutical composition and the carrier is a pharmaceuticallyacceptable carrier. The glucagon analogue peptide may be in the form ofa pharmaceutically acceptable salt of the glucagon analogue.

The compounds described herein find use, inter alia, in preventingweight gain or promoting weight loss. By “preventing” is meantinhibiting or reducing when compared to the absence of treatment, and isnot necessarily meant to imply complete cessation of weight gain. Thepeptides may cause a decrease in food intake and/or increased energyexpenditure, resulting in the observed effect on body weight.Independently of their effect on body weight, the compounds of theinvention may have a beneficial effect on glucose control and/or oncirculating cholesterol levels, being capable of lowering circulatingLDL levels and increasing HDL/LDL ratio. Thus the compounds of theinvention can be used for direct or indirect therapy of any conditioncaused or characterised by excess body weight, such as the treatmentand/or prevention of obesity, morbid obesity, obesity linkedinflammation, obesity linked gallbladder disease, obesity induced sleepapnea. They may also be used for the prevention of conditions caused orcharacterised by inadequate glucose control or dyslipidaemia (e.g.elevated LDL levels or reduced HDL/LDL ratio), diabetes (especially Type2 diabetes), metabolic syndrome, hypertension, atherogenic dyslipidemia,atherosclerosis, arteriosclerosis, coronary heart disease, peripheralartery disease, stroke or microvascular disease. Their effects in theseconditions may be as a result of or associated with their effect on bodyweight, or may be independent thereof.

The invention also provides a compound of the invention for use in amethod of medical treatment, particularly for use in a method oftreatment of a condition as described above.

The invention also provides the use of a compound of the invention inthe preparation of a medicament for the treatment of a condition asdescribed above.

The compound of the invention may be administered as part of acombination therapy with an agent for treatment of diabetes, obesity,dyslipidaemia or hypertension.

In such cases, the two active agents may be given together orseparately, and as part of the same pharmaceutical formulation or asseparate formulations.

Thus the compound of the invention can be used in combination with ananti-diabetic agent including but not limited to a biguanide (e.g.metformin), a sulfonylurea, a meglitinide or glinide (e.g. nateglinide),a DPP-IV inhibitor, a glitazone, an insulin, or an insulin analogue.Examples of insulin analogues include but are not limited to Lantus™,Novorapid™, Humalog™, Novomix™, Actraphane HM™, Levemir™ and Apidra™.

The compound can further be used in combination with an anti-obesityagent including but not limited to a glucagon-like peptide receptor 1agonist, peptide YY or analogue thereof, cannabinoid receptor 1antagonist, lipase inhibitor, melanocortin receptor 4 agonist, ormelanin concentrating hormone receptor 1 antagonist.

The compound can further be used in combination with ananti-hypertension agent including but not limited to anangiotensin-converting enzyme inhibitor, angiotensin II receptorblocker, diuretic, beta-blocker, or calcium channel blocker.

The compound can be used in combination with an anti-dyslipidaemia agentincluding but not limited to a statin, a fibrate, a niacin or acholesterol absorbtion inhibitor.

Thus the invention further provides a composition or therapeutic kitcomprising a compound of the invention and for example an anti-diabeticagent, anti-obesity agent, anti-hypertension agent or anti-dyslipidaemiaagent as described above. Also provided is such a composition ortherapeutic kit for use in a method of medical treatment, especially fortreatment of a condition as described above.

The compound of the invention may be made by synthetic chemistry.Accordingly the invention provides a method of synthesis of a compoundof the invention.

As already described, the invention extends to nucleic acids encodingthe peptide sequence X or X—Z, as well as expression vectors comprisingthe above-described nucleic acid sequence (optionally operably linked tosequences to direct its expression) and host cells containing thenucleic acids or expression vectors. Preferably the host cells arecapable of expressing and optionally secreting the compound of theinvention.

The present invention provides a method of producing a compound of theinvention, the method comprising culturing the host cells underconditions suitable for expressing the peptide sequence X or X—Z andpurifying the compound thus produced. This is particularly useful wherethe peptide contains only naturally-occurring amino acids.

Where the compound of the invention contains one or morenon-naturally-occurring amino acids, the method may comprise expressinga peptide sequence containing one or more differences from the sequenceX or X—Z, optionally purifying the compound thus produced, and adding ormodifying one or more amino acids to produce a compound of the inventionor a compound comprising the amino acid sequence X or X—Z.

Whichever method is used to produce the compound of the invention, itmay comprise one or more further steps of modifying the sequence X orX—Z, especially to introduce one or more lipophilic and/or polymericmoieties as defined elsewhere in this specification.

The invention further provides a nucleic acid of the invention, anexpression vector of the invention, or a host cell capable of expressingand optionally secreting a compound of the invention, for use in amethod of medical treatment. It will be understood that the nucleicacid, expression vector and host cells may be used for treatment of anyof the disorders described herein which may be treated with thecompounds of the invention themselves. References to a therapeuticcomposition comprising a compound of the invention, administration of acompound of the invention, or any therapeutic use thereof, shouldtherefore be construed to encompass the equivalent use of a nucleicacid, expression vector or host cell of the invention, except where thecontext demands otherwise.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification, the conventional one letter and threeletter codes for naturally occurring amino acids are used, as well asgenerally accepted three letter codes for other amino acids, such as Aib(α-aminoisobutyric acid), Hse (homoserine), Orn (ornithine), Dbu(2,4-diaminobutyric acid), Dpr (2,3-diaminopropanoic acid).

Glucagon is a 29-amino acid peptide that corresponds to amino acids 53to 81 of pre-proglucagon and has the sequenceHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-TyrLeu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(SEQ ID NO: 26). Oxyntomodulin (OXM) is a 37 amino acid peptide whichincludes the complete 29 amino acid sequence of glucagon with anoctapeptide carboxyterminal extension (amino acids 82 to 89 ofpre-proglucagon, having the sequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala(SEQ ID NO: 27) and termed “intervening peptide 1” or IP-1; the fullsequence of human oxyntomodulin is thusHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-le-Ala)(SEQ ID NO: 28). The major biologically active fragment of GLP-1 isproduced as a 30-amino acid, C-terminally amidated peptide thatcorresponds to amino acids 98 to 127 of pre-proglucagon.

The term “native glucagon” thus refers to native human glucagon havingthe sequenceH-HisSer-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH(SEQ ID NO: 26).

Amino acids within the sequence X of the compounds of the invention canbe considered to be numbered consecutively from 1 to 29 in theconventional N-terminal to C-terminal direction. Reference to a“position” within X should be construed accordingly, as should referenceto positions within native human glucagon and other molecules.

A compound of the invention may comprise a C-terminal peptide sequence Zof 1-20 amino acids, for example to stabilise the conformation and/orsecondary structure of the glucagon analogue peptide, and/or to renderthe glucagon analogue peptide more resistant to enzymatic hydrolysis,e.g. as described in WO99/46283.

When present, Z represents a peptide sequence of 1-20 amino acidresidues, e.g. in the range of 1-15, more preferably in the range of1-10, in particular in the range of 1-7 amino acid residues, e.g., 1, 2,3, 4, 5, 6 or 7 amino acid residues, such as 6 amino acid residues. Eachof the amino acid residues in the peptide sequence Z may independentlybe selected from Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu(2,4-diaminobutyric acid), Dpr (2,3-diaminopropanoic acid) and Orn(ornithine). Preferably, the amino acid residues are selected from Ser,Thr, Tyr, Glu, Lys, Arg, Dbu, Dpr and Orn, more preferably selectedexclusively from Glu, Lys, and Cys. The above-mentioned amino acids mayhave either D- or L-configuration, which in certain embodiments, have anL-configuration. Particularly preferred sequences Z are sequences offour, five, six or seven consecutive lysine residues (i.e. Lys₃, Lys₄,Lys₅, Lys₆ or Lys₇), and particularly five or six consecutive lysineresidues. Other exemplary sequences of Z are shown in WO 01/04156.Alternatively the C-terminal residue of the sequence Z may be a Cysresidue. This may assist in modification (e.g. PEGylation, orconjugation to albumin) of the compound. In such embodiments, thesequence Z may, for example, be only one amino acid in length (i.e.Z=Cys) or may be two, three, four, five, six or even more amino acids inlength. The other amino acids therefore serve as a spacer between thepeptide X and the terminal Cys residue.

The peptide sequence Z has no more than 25% sequence identity with thecorresponding sequence of the IP-1 portion of human OXM (which has thesequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala).

“Percent (%) amino acid sequence identity” of a given peptide orpolypeptide sequence with respect to another polypeptide sequence (e.g.IP-1) is calculated as the percentage of amino acid residues in thegiven peptide sequence that are identical with correspondinglypositioned amino acid residues in the corresponding sequence of thatother polypeptide when the two are aligned with one another, introducinggaps for optimal alignment if necessary. % identity values may bedetermined using WU-BLAST-2 (Altschul et al., Methods in Enzymology,266:460-480 (1996)). WU-BLAST-2 uses several search parameters, most ofwhich are set to the default values. The adjustable parameters are setwith the following values: overlap span=1, overlap fraction=0.125, wordthreshold (T)=11. A % amino acid sequence identity value is determinedby the number of matching identical residues as determined byWU-BLAST-2, divided by the total number of residues of the referencesequence (gaps introduced by WU-BLAST-2 into the reference sequence tomaximize the alignment score being ignored), multiplied by 100.

Thus, when Z is aligned optimally with the 8 amino acids of IP-1, it hasno more than two amino acids which are identical with the correspondingamino acids of IP-1.

In certain embodiments, Z is absent.

One or more of the amino acid side chains in the compound of theinvention may be conjugated to a lipophilic substituent. The lipophilicsubstituent may be covalently bonded to an atom in the amino acid sidechain, or alternatively may be conjugated to the amino acid side chainby a spacer. A lipophilic substituent may be conjugated to a side chainof an amino acid which is part of the peptide X, and/or to a side chainof an amino acid which is part of the peptide Z.

Without wishing to be bound by any particular theory, it is thought thatthe lipophilic substituent binds albumin in the blood stream, thusshielding the compounds of the invention from enzymatic degradation andthereby enhancing the half-life of the compounds. It may also modulatethe potency of the compound, e.g. with respect to the glucagon receptorand/or the GLP-1 receptor.

In certain embodiments, only one amino acid side chain is conjugated toa lipophilic substituent. In other embodiments, two amino acid sidechains are each conjugated to a lipophilic substituent. In yet furtherembodiments, three or even more amino acid side chains are eachconjugated to a lipophilic substituent. When a compound contains two ormore lipophilic substituents, they may be the same or different.

The lipophilic substituent may comprise or consist of a lipophilicmoiety Z¹ which may be covalently bonded directly to an atom in theamino acid side chain, or alternatively may be conjugated to the aminoacid side chain by a spacer Z².

The term “conjugated” is used here to describe the physical attachmentof one identifiable chemical moiety to another, and the structuralrelationship between such moieties. It should not be taken to imply anyparticular method of synthesis.

The lipophilic moiety may be attached to the amino acid side chain or tothe spacer via an ester, a sulphonyl ester, a thioester, an amide, acarbamate, a urea or a sulphonamide. Accordingly it will be understoodthat preferably the lipophilic substituent includes an acyl group, asulphonyl group, an N atom, an O atom or an S atom which forms part ofthe ester, sulphonyl ester, thioester, amide or sulphonamide.Preferably, an acyl group in the lipophilic substituent forms part of anamide or ester with the amino acid side chain or the spacer.

The lipophilic moiety may include a hydrocarbon chain having 4 to 30 Catoms. Preferably it has at least 8 or 12 C atoms, and preferably it has24 C atoms or fewer, or 20 C atoms or fewer. The hydrocarbon chain maybe linear or branched and may be saturated or unsaturated. It will beunderstood that the hydrocarbon chain is preferably substituted with amoiety which forms part of the attachment to the amino acid side chainor the spacer, for example an acyl group, a sulphonyl group, an N atom,an O atom or an S atom. Most preferably the hydrocarbon chain issubstituted with acyl, and accordingly the hydrocarbon chain may be partof an alkanoyl group, for example palmitoyl, caproyl, lauroyl, myristoylor stearoyl.

Accordingly, the lipophilic moiety may have the formula shown below:

A may be, for example, an acyl group, a sulphonyl group, NH, N-alkyl, anO atom or an S atom, preferably acyl. n is an integer from 3 to 29,preferably from 7 to 25, more preferred 11 to 21, even more preferred 15to 19.

The hydrocarbon chain may be further substituted. For example, it may befurther substituted with up to three substituents selected from NH₂, OHand COOH, especially at the free end of the molecule distal from thespacer or peptide. For example, it may comprise a free carboxylic acidgroup.

If the hydrocarbon chain is further substituted, preferably it isfurther substituted with only one substituent. Alternatively oradditionally, the hydrocarbon chain may include a cycloalkane orheterocycloalkane, for example as shown below:

Preferably the cycloalkane or heterocycloalkane is a six-membered ring.Most preferably, it is piperidine.

Alternatively, the lipophilic moiety may be based on acyclopentanophenanthrene skeleton, which may be partially or fullyunsaturated, or saturated. The carbon atoms in the skeleton each may besubstituted with Me or OH. For example, the lipophilic substituent maybe cholyl, deoxycholyl or lithocholyl.

As mentioned above, the lipophilic moiety may be conjugated to the aminoacid side chain by a spacer. When present, the spacer is attached to thelipophilic moiety and to the amino acid side chain. The spacer may beattached to the lipophilic moiety and to the amino acid side chainindependently by an ester, a sulphonyl ester, a thioester, an amide, acarbamate, a urea or a sulphonamide. Accordingly, it may include twomoieties independently selected from acyl, sulphonyl, an N atom, an Oatom or an S atom. The spacer may have the formula:

wherein B and D are each independently selected from acyl, sulphonyl,NH, N-alkyl, an O atom and an S atom, preferably from acyl and NH.Preferably, n is an integer from 1 to 10, preferably from 1 to 5. Thespacer may be further substituted with one or more substituents selectedfrom C₀₋₆ alkyl, C₀₋₆ alkyl amine, C₀₋₆ alkyl hydroxy and C₀₋₆ alkylcarboxy.

Alternatively, the spacer may have two or more repeat units of theformula above. B, D and n are each selected independently for eachrepeat unit. Adjacent repeat units may be covalently attached to eachother via their respective B and D moieties. For example, the B and Dmoieties of the adjacent repeat units may together form an ester, asulphonyl ester, a thioester, an amide or a sulphonamide. The free B andD units at each end of the spacer are attached to the amino acid sidechain and the lipophilic moiety as described above.

Preferably the spacer has five or fewer, four or fewer or three or fewerrepeat units. Most preferably the spacer has two repeat units, or is asingle unit.

The spacer (or one or more of the repeat units of the spacer, if it hasrepeat units) may be, for example, a natural or unnatural amino acid. Itwill be understood that for amino acids having functionalised sidechains, B and/or D may be a moiety within the side chain of the aminoacid. The spacer may be any naturally occurring or unnatural amino acid.For example, the spacer (or one or more of the repeat units of thespacer, if it has repeat units) may be Gly, Pro, Ala, Val, Leu, lie,Met, Cys, Phe, Tyr, Trp, His, Lys, Arg, Gln, Asn, α-Glu, γ-Glu, Asp, SerThr, Gaba, Aib, β-Ala, 5-aminopentanoyl, 6-aminohexanoyl,7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl or 10-aminodecanoyl.

For example, the spacer may be a single amino acid selected from γ-Glu,Gaba, β-Ala and α-Glu.

A lipophilic substituent may be conjugated to any amino acid side chainin a compound of the invention. Preferably, the amino acid side chainincludes a carboxy, hydroxyl, thiol, amide or amine group, for formingan ester, a sulphonyl ester, a thioester, an amide or a sulphonamidewith the spacer or lipophilic substituent. For example, the lipophilicsubstituent may be conjugated to Asn, Asp, Glu, Gln, His, Lys, Arg, Ser,Thr, Tyr, Trp, Cys or Dbu, Dpr or Orn. Preferably, the lipophilicsubstituent is conjugated to Lys. An amino acid shown as Lys in theformulae provided herein may be replaced by, e.g., Dbu, Dpr or Orn wherea lipophilic substituent is added.

An example of a lipophilic substituent comprising lipophilic moiety andspacer is shown in the formula below:

Here, a Lys residue in the compound of the present invention iscovalently attached to γ-Glu (the spacer) via an amide moiety. Palmitoyl(i.e. hexadecanoyl) is covalently attached to the γ-Glu spacer via anamide moiety, thus creating a hexadecanoyl-isoGlu group.

Alternatively or additionally, one or more amino acid side chains in thecompound of the invention may be conjugated to a polymeric moiety, forexample, in order to increase solubility and/or half-life in vivo (e.g.in plasma) and/or bioavailability. Such modification is also known toreduce clearance (e.g. renal clearance) of therapeutic proteins andpeptides.

The skilled reader will be well aware of suitable techniques that can beused to perform the coupling reactions with spacer and lipophilic moietyusing general synthetic methodology listed e.g. in “ComprehensiveOrganic Transformations, A Guide to Functional Group Preparations”, 2ndedition, Larock, R. C.; Wiley-VCH: New York, 1999. Such transformationsmay take place at any suitable stage during the synthesis process.

The polymeric moiety is preferably water-soluble (amphiphilic orhydrophilic), non-toxic, and pharmaceutically inert. Suitable polymericmoieties include polyethylene glycol (PEG), homo- or co-polymers of PEG,a monomethyl-substituted polymer of PEG (mPEG), and polyoxyethyleneglycerol (POG). See, for example, Int. J. Hematology 68:1 (1998);Bioconjugate Chem. 6:150 (1995); and Crit. Rev. Therap. Drug CarrierSys. 9:249 (1992).

Other suitable polymeric moieties include poly-amino acids such aspoly-lysine, poly-aspartic acid and poly-glutamic acid (see for exampleGombotz, et al. (1995), Bioconjugate Chem., vol. 6: 332-351; Hudecz, etal. (1992), Bioconjugate Chem., vol. 3, 49-57; Tsukada, et al. (1984),J. Natl. Cancer Inst., vol 73, 721-729; and Pratesi, et al. (1985), Br.J. Cancer, vol. 52: 841-848).

The polymeric moiety may be straight-chain or branched. It may have amolecular weight of 500-40,000 Da, for example 500-10,000 Da, 1000-5000Da, 10,000-20,000 Da, or 20,000-40,000 Da.

A compound of the invention may comprise two or more such moieties, inwhich case the total molecular weight of all such moieties willgenerally fall within the ranges provided above.

The polymeric moiety may be coupled (by covalent linkage) to an amino,carboxyl or thiol group of an amino acid side chain. Preferred examplesare the thiol group of Cys residues and the epsilon amino group of Lysresidues. The carboxyl groups of Asp and Glu residues may also be used.

The skilled reader will be well aware of suitable techniques that can beused to perform the coupling reaction. For example, a PEG moietycarrying a methoxy group can be coupled to a Cys thiol group by amaleimido linkage using reagents commercially available from NektarTherapeutics. See also WO 2008/101017, and the references cited above,for details of suitable chemistry.

Peptide Synthesis

The compounds of the present invention may be manufactured either bystandard synthetic methods, recombinant expression systems, or any otherstate of the art method. Thus the glucagon analogues may be synthesizedin a number of ways, including, for example, a method which comprises:

(a) synthesizing the peptide by means of solid-phase or liquid-phasemethodology, either stepwise or by fragment assembly, and isolation andpurifying of the final peptide product; or

(b) expressing a nucleic acid construct that encodes the peptide in ahost cell, and recovering the expression product from the host cell orculture medium; or

(c) effecting cell-free in vitro expression of a nucleic acid constructthat encodes the peptide, and recovering the expression product;

or any combination of methods of (a), (b), and (c) to obtain fragmentsof the peptide, subsequently ligating the fragments to obtain thepeptide, and recovering the peptide.

It is preferred to synthesize the analogues of the invention by means ofsolid-phase or liquid-phase peptide synthesis. In this context,reference is made to WO 98/11125 and, among many others, Fields, G B etal., 2002, “Principles and practice of solid-phase peptide synthesis”.In: Synthetic Peptides (2nd Edition), and the Examples herein.

For recombinant expression, the nucleic acid fragments of the inventionwill normally be inserted in suitable vectors to form cloning orexpression vectors carrying the nucleic acid fragments of the invention;such novel vectors are also part of the invention. The vectors can,depending on purpose and type of application, be in the form ofplasmids, phages, cosmids, mini-chromosomes, or virus, but also nakedDNA which is only expressed transiently in certain cells is an importantvector. Preferred cloning and expression vectors (plasmid vectors) ofthe invention are capable of autonomous replication, thereby enablinghigh copy-numbers for the purposes of high-level expression orhigh-level replication for subsequent cloning.

In general outline, an expression vector comprises the followingfeatures in the 5′→3′ direction and in operable linkage: a promoter fordriving expression of the nucleic acid fragment of the invention,optionally a nucleic acid sequence encoding a leader peptide enablingsecretion (to the extracellular phase or, where applicable, into theperiplasma), the nucleic acid fragment encoding the peptide of theinvention, and optionally a nucleic acid sequence encoding a terminator.They may comprise additional features such as selectable markers andorigins of replication. When operating with expression vectors inproducer strains or cell lines it may be preferred that the vector iscapable of integrating into the host cell genome. The skilled person isvery familiar with suitable vectors and is able to design one accordingto their specific requirements.

The vectors of the invention are used to transform host cells to producethe compound of the invention. Such transformed cells, which are alsopart of the invention, can be cultured cells or cell lines used forpropagation of the nucleic acid fragments and vectors of the invention,or used for recombinant production of the peptides of the invention.

Preferred transformed cells of the invention are micro-organisms such asbacteria [such as the species Escherichia (e.g. E. coli), Bacillus (e.g.Bacillus subtilis), Salmonella, or Mycobacterium (preferablynon-pathogenic, e.g. M. bovis BCG), yeasts (e.g., Saccharomycescerevisiae and Pichia pastoris), and protozoans. Alternatively, thetransformed cells may be derived from a multicellular organism, i.e. itmay be fungal cell, an insect cell, an algal cell, a plant cell, or ananimal cell such as a mammalian cell. For the purposes of cloning and/oroptimised expression it is preferred that the transformed cell iscapable of replicating the nucleic acid fragment of the invention. Cellsexpressing the nucleic fragment are useful embodiments of the invention;they can be used for small-scale or large-scale preparation of thepeptides of the invention.

When producing the peptide of the invention by means of transformedcells, it is convenient, although far from essential, that theexpression product is secreted into the culture medium.

Efficacy

Binding of the relevant compounds to GLP-1 or glucagon (Glu) receptorsmay be used as an indication of agonist activity, but in general it ispreferred to use a biological assay which measures intracellularsignalling caused by binding of the compound to the relevant receptor.For example, activation of the glucagon receptor by a glucagon agonistwill stimulate cellular cyclic AMP (cAMP) formation. Similarly,activation of the GLP-1 receptor by a GLP-1 agonist will stimulatecellular cAMP formation. Thus, production of cAMP in suitable cellsexpressing one of these two receptors can be used to monitor therelevant receptor activity. Use of a suitable pair of cell types, eachexpressing one receptor but not the other, can hence be used todetermine agonist activity towards both types of receptor.

The skilled person will be aware of suitable assay formats, and examplesare provided below. The GLP-1 receptor and/or the glucagon receptor mayhave the sequence of the receptors as described in the examples. Forexample, the assays may employ the human glucagon receptor (Glucagon-R)having primary accession number GI:4503947 and/or the humanglucagon-like peptide 1 receptor (GLP-1R) having primary accessionnumber GI:166795283. (in that where sequences of precursor proteins arereferred to, it should of course be understood that assays may make useof the mature protein, lacking the signal sequence).

EC₅₀ values may be used as a numerical measure of agonist potency at agiven receptor. An EC₅₀ value is a measure of the concentration of acompound required to achieve half of that compound's maximal activity ina particular assay. Thus, for example, a compound having EC₅₀[GLP-1]lower than the EC₅₀[GLP-1] of glucagon in a particular assay may beconsidered to have higher GLP-1 receptor agonist potency than glucagon.

The compounds described in this specification are typically GluGLP-1dual agonists, as determined by the observation that they are capable ofstimulating cAMP formation at both the glucagon receptor and the GLP-1receptor. The stimulation of each receptor can be measured inindependent assays and afterwards compared to each other.

By comparing the EC₅₀ value for the GLP-1 receptor (EC₅₀ [GLP-1-R]) withthe EC₅₀ value for the Glucagon receptor, (EC₅₀ [GlucagonR]) for a givencompound. the relative GLP-1R selectivity can be calculated as follows:Relative GLP-1R selectivity [compound]=(EC₅₀ [GLP-1R])/(EC₅₀[Glucagon-R])

The term “EC₅₀” stands for the half maximal Effective Concentration,typically at a particular receptor, or on the level of a particularmarker for receptor function, and can refer to an inhibitory or anantagonistic activity, depending on the specific biochemical context.

Without wishing to be bound by any particular theory, a compound'srelative selectivity may allow its effect on the GLP-1 or glucagonreceptor to be compared directly to its effect on the other receptor.For example, the higher a compound's relative GLP-1 selectivity is, themore effective that compound may be on the GLP-1 receptor as compared tothe glucagon receptor. Typically the results are compared for glucagonand GLP-1 receptors from the same species, e.g. human glucagon and GLP-1receptors, or murine glucagon and GLP-1 receptors.

The compounds of the invention may have a higher relative GLP-1Rselectivity than human glucagon in that for a particular level ofglucagon-R agonist activity, the compound may display a higher level ofGLP-1R agonist activity (i.e. greater potency at the GLP-1 receptor)than glucagon. It will be understood that the absolute potency of aparticular compound at the glucagon and GLP-1 receptors may be higher,lower or approximately equal to that of native human glucagon, as longas the appropriate relative GLP-1R selectivity is achieved.

Nevertheless, the compounds of this invention may have a lower EC₅₀[GLP-1R] than human glucagon. The compounds may have a lowerEC₅₀[GLP-1-R] than glucagon while maintaining an EC₅₀ [Glucagon-R] thatis less than 10-fold higher than that of human glucagon, less than5-fold higher than that of human glucagon, or less than 2-fold higherthan that of human glucagon.

The compounds of the invention may have an EC₅₀ [Glucagon-R] that isless than two-fold that of human glucagon. The compounds may have anEC₅₀ [Glucagon-R] that is less than two-fold that of human glucagon andhave an EC₅₀ [GLP-1R] that is less than half that of human glucagon,less than a fifth of that of human glucagon, or less than a tenth ofthat of human glucagon.

The relative GLP-1R selectivity of the compounds may be between 0.05 and20. For example, the compounds may have a relative selectivity of0.05-0.20, 0.1-0.30, 0.2-0.5, 0.3-0.7, or 0.5-1.0; 1.0-2.0, 1.5-3.0,2.0-4.0 or 2.5-5.0; or 0.05-20, 0.075-15, 0.1-10, 0.15-5, 0.75-2.5 or0.9-1.1.

In certain embodiments, it may be desirable that EC₅₀ of any givencompound for both the Glucagon-R and GLP-1R, e.g. for the human glucagonand GLP-1 receptors, should be less than 1 nM.

Therapeutic Uses

The compounds of the invention may provide attractive treatment and/orprevention options for, inter alia, obesity and metabolic diseasesincluding diabetes, as discussed below.

Diabetes comprises a group of metabolic diseases characterized byhyperglycemia resulting from defects in insulin secretion, insulinaction, or both. Acute signs of diabetes include excessive urineproduction, resulting compensatory thirst and increased fluid intake,blurred vision, unexplained weight loss, lethargy, and changes in energymetabolism. The chronic hyperglycemia of diabetes is associated withlong-term damage, dysfunction, and failure of various organs, notablythe eyes, kidneys, nerves, heart and blood vessels. Diabetes isclassified into type 1 diabetes, type 2 diabetes and gestationaldiabetes on the basis on pathogenetic characteristics.

Type 1 diabetes accounts for 5-10% of all diabetes cases and is causedby auto-immune destruction of insulin-secreting pancreatic β-cells.

Type 2 diabetes accounts for 90-95% of diabetes cases and is a result ofa complex set of metabolic disorders. Type 2 diabetes is the consequenceof endogenous insulin production becoming insufficient to maintainplasma glucose levels below the diagnostic thresholds.

Gestational diabetes refers to any degree of glucose intoleranceidentified during pregnancy.

Pre-diabetes includes impaired fasting glucose and impaired glucosetolerance and refers to those states that occur when blood glucoselevels are elevated but below the levels that are established for theclinical diagnosis for diabetes.

A large proportion of people with type 2 diabetes and pre-diabetes areat increased risk of morbidity and mortality due to the high prevalenceof additional metabolic risk factors including abdominal obesity(excessive fat tissue around the abdominal internal organs), atherogenicdyslipidemia (blood fat disorders including high triglycerides, low HDLcholesterol and/or high LDL cholesterol, which foster plaque buildup inartery walls), elevated blood pressure (hypertension) a prothromboticstate (e.g. high fibrinogen or plasminogen activator inhibitor-1 in theblood), and proinflammatory state (e.g., elevated C-reactive protein inthe blood).

Conversely, obesity confers an increased risk of developingpre-diabetes, type 2 diabetes as well as e.g. certain types of cancer,obstructive sleep apnea and gall-bladder disease.

Dyslipidaemia is associated with increased risk of cardiovasculardisease. High Density Lipoprotein (HDL) is of clinical importance sincean inverse correlation exists between plasma HDL concentrations and riskof atherosclerotic disease. The majority of cholesterol stored inatherosclerotic plaques originates from LDL and hence elevatedconcentrations Low Density Lipoproteins (LDL) is closely associated withatherosclerosis. The HDL/LDL ratio is a clinical risk indictor foratherosclerosis and coronary atherosclerosis in particular.

Metabolic syndrome is characterized by a group of metabolic risk factorsin one person. They include abdominal obesity (excessive fat tissuearound the abdominal internal organs), atherogenic dyslipidemia (bloodfat disorders including high triglycerides, low HDL cholesterol and/orhigh LDL cholesterol, which foster plaque buildup in artery walls),elevated blood pressure (hypertension), insulin resistance and glucoseintolerance, prothrombotic state (e.g. high fibrinogen or plasminogenactivator inhibitor-1 in the blood), and proinflammatory state (e.g.,elevated C-reactive protein in the blood).

Individuals with the metabolic syndrome are at increased risk ofcoronary heart disease and other diseases related to othermanifestations of arteriosclerosis (e.g., stroke and peripheral vasculardisease). The dominant underlying risk factors for this syndrome appearto be abdominal obesity.

Without wishing to be bound by any particular theory, it is believedthat the compounds of the invention act as dual agonists both on thehuman glucagon-receptor and the human GLP1-receptor, abbreviated here asdual GluGLP-1 agonists. The dual agonist may combine the effect ofglucagon, e.g. on fat metabolism, with the effect of GLP-1, e.g. onblood glucose levels and food intake. They may therefore act toaccelerate elimination of excessive adipose tissue, induce sustainableweight loss, and improve glycaemic control. Dual GluGLP-1 agonists mayalso act to reduce cardiovascular risk factors such as high cholesterol,high LDL-cholesterol or low HDL/LDL cholesterol ratios.

The compounds of the present invention can therefore be used in asubject in need thereof as pharmaceutical agents for preventing weightgain, promoting weight loss, reducing excess body weight or treatingobesity (e.g. by control of appetite, feeding, food intake, calorieintake, and/or energy expenditure), including morbid obesity, as well asassociated diseases and health conditions including but not limited toobesity linked inflammation, obesity linked gallbladder disease andobesity induced sleep apnea. The compounds of the invention may also beused for treatment of conditions caused by or associated with impairedglucose control, including metabolic syndrome, insulin resistance,glucose intolerance, pre-diabetes, increased fasting glucose, type 2diabetes, hypertension, atherosclerois, arteriosclerosis, coronary heartdisease, peripheral artery disease and stroke, in a subject in needthereof. Some of these conditions can be associated with obesity.However, the effects of the compounds of the invention on theseconditions may be mediated in whole or in part via an effect on bodyweight, or may be independent thereof.

The synergistic effect of dual GluGLP-1 agonists may also result inreduction of cardiovascular risk factors such as high cholesterol andLDL, which may be entirely independent of their effect on body weight.

Thus the invention provides the use of a compound of the invention inthe treatment of a condition as described above, in an individual inneed thereof.

The invention also provides a compound of the invention for use in amethod of medical treatment, particularly for use in a method oftreatment of a condition as described above.

In a preferred aspect, the compounds described may be used in treatingdiabetes, esp. type 2 diabetes.

In a specific embodiment, the present invention comprises use of acompound for treating diabetes, esp. type 2 diabetes in an individual inneed thereof.

In a not less preferred aspect, the compounds described may be used inpreventing weight gain or promoting weight loss.

In a specific embodiment, the present invention comprises use of acompound for preventing weight gain or promoting weight loss in anindividual in need thereof.

In a specific embodiment, the present invention comprises use of acompound in a method of treatment of a condition caused or characterisedby excess body weight, e.g. the treatment and/or prevention of obesity,morbid obesity, morbid obesity prior to surgery, obesity linkedinflammation, obesity linked gallbladder disease, obesity induced sleepapnea, prediabetes, diabetes, esp. type 2 diabetes, hypertension,atherogenic dyslipidimia, atherosclerois, arteriosclerosis, coronaryheart disease, peripheral artery disease, stroke or microvasculardisease in an individual in need thereof.

In another aspect, the compounds described may be used in a method oflowering circulating LDL levels, and/or increasing HDL/LDL ratio.

In a specific embodiment, the present invention comprises use of acompound in a method of lowering circulating LDL levels, and/orincreasing HDL/LDL ratio in an individual in need thereof.

In another aspect, the compounds described may be used in a method oflowering circulating triglyceride levels.

Pharmaceutical Compositions

The compounds of the present invention may be formulated aspharmaceutical compositions prepared for storage or administration. Sucha composition typically comprises a therapeutically effective amount ofa compound of the invention, in the appropriate form, in apharmaceutically acceptable carrier.

The therapeutically effective amount of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific mammalunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy, and may depend on such factors as weight, diet,concurrent medication and other factors, well known to those skilled inthe medical arts. The dosage sizes and dosing regimen most appropriatefor human use may be guided by the results obtained by the presentinvention, and may be confirmed in properly designed clinical trials.The compounds of the present invention may be particularly useful fortreatment of humans.

An effective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

The term “pharmaceutically acceptable carrier” includes any of thestandard pharmaceutical carriers. Pharmaceutically acceptable carriersfor therapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro edit. 1985). For example, sterile salineand phosphate-buffered saline at slightly acidic or physiological pH maybe used. pH buffering agents may be phosphate, citrate, acetate,tris/hydroxymethyl)aminomethane (TRIS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS),ammonium bicarbonate, diethanolamine, histidine, which is a preferredbuffer, arginine, lysine, or acetate or mixtures thereof. The termfurther encompasses any agents listed in the US Pharmacopeia for use inanimals, including humans.

The term “pharmaceutically acceptable salt” refers to a salt of any oneof the compounds of the invention. Salts include pharmaceuticallyacceptable salts such as acid addition salts and basic salts. Examplesof acid addition salts include hydrochloride salts, citrate salts andacetate salts. Examples of basic salts include salts where the cation isselected from alkali metals, such as sodium and potassium, alkalineearth metals, such as calcium, and ammonium ions ⁺N(R³)₃(R⁴), where R³and R⁴ independently designates optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substituted aryl, oroptionally substituted heteroaryl. Other examples of pharmaceuticallyacceptable salts are described in “Remington's Pharmaceutical Sciences”,17th edition. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company,Easton, Pa., U.S.A., 1985 and more recent editions, and in theEncyclopaedia of Pharmaceutical Technology.

“Treatment” is an approach for obtaining beneficial or desired clinicalresults. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “Treatment” is an intervention performed with theintention of preventing the development or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures in certain embodiments. Thosein need of treatment include those already with the disorder as well asthose in which the disorder is to be prevented. By treatment is meantinhibiting or reducing an increase in pathology or symptoms (e.g. weightgain, hyperglycemia) when compared to the absence of treatment, and isnot necessarily meant to imply complete cessation of the relevantcondition.

The pharmaceutical compositions can be in unit dosage form. In suchform, the composition is divided into unit doses containing appropriatequantities of the active component. The unit dosage form can be apackaged preparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms. It may be provided in single dose injectable form, forexample in the form of a pen. In certain embodiments, packaged formsinclude a label or insert with instructions for use. Compositions may beformulated for any suitable route and means of administration.Pharmaceutically acceptable carriers or diluents include those used informulations suitable for oral, rectal, nasal, topical (including buccaland sublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, and transdermal)administration. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy.

Subcutaneous or transdermal modes of administration may be particularlysuitable for the compounds described herein.

Compositions of the invention may further be compounded in, or attachedto, for example through covalent, hydrophobic and electrostaticinteractions, a drug carrier, drug delivery system and advanced drugdelivery system in order to further enhance stability of the compound,increase bioavailability, increase solubility, decrease adverse effects,achieve chronotherapy well known to those skilled in the art, andincrease patient compliance or any combination thereof. Examples ofcarriers, drug delivery systems and advanced drug delivery systemsinclude, but are not limited to, polymers, for example cellulose andderivatives, polysaccharides, for example dextran and derivatives,starch and derivatives, poly(vinyl alcohol), acrylate and methacrylatepolymers, polylactic and polyglycolic acid and block co-polymersthereof, polyethylene glycols, carrier proteins, for example albumin,gels, for example, thermogelling systems, for example block co-polymericsystems well known to those skilled in the art, micelles, liposomes,microspheres, nanoparticulates, liquid crystals and dispersions thereof,L2 phase and dispersions there of, well known to those skilled in theart of phase behaviour in lipid-water systems, polymeric micelles,multiple emulsions, self-emulsifying, self-microemulsifying,cyclodextrins and derivatives thereof, and dendrimers.

Combination Therapy

A compound or composition of the invention may be administered as partof a combination therapy with an agent for treatment of obesity,hypertension, dyslipidemia or diabetes.

In such cases, the two active agents may be given together orseparately, and as part of the same pharmaceutical formulation or asseparate formulations.

Thus a compound or composition of the invention can further be used incombination with an anti-obesity agent, including but not limited to aglucagon-like peptide receptor 1 agonist, peptide YY or analoguethereof, cannabinoid receptor 1 antagonist, lipase inhibitor,melanocortin receptor 4 agonist, or melanin concentrating hormonereceptor 1 antagonist.

A compound or composition of the invention can be used in combinationwith an anti-hypertension agent, including but not limited to anangiotensin-converting enzyme inhibitor, angiotensin II receptorblocker, diuretics, beta-blocker, or calcium channel blocker.

A compound or composition of the invention can be used in combinationwith a dyslipidaemia agent, including but not limited to a statin, afibrate, a niacin and/or a cholesterol absorbtion inhibitor.

Further, a compound or composition of the invention can be used incombination with an anti-diabetic agent, including but not limited to abiguanide (e.g. metformin), a sulfonylurea, a meglitinide or glinide(e.g. nateglinide), a DPP-IV inhibitor, a glitazone, a different GLP-1agonist, an insulin or an insulin analogue. In a preferred embodiment,the compound or salt thereof is used in combination with insulin or aninsulin analogue, DPP-IV inhibitor, sulfonylurea or metformin,particularly sulfonylurea or metformin, for achieving adequate glycemiccontrol. Examples of insulin analogues include but are not limited toLantus, Novorapid, Humalog, Novomix, and Actraphane HM, Levemir andApidra.

EXAMPLES Example 1: General Synthesis of Glucagon Analogues

Solid phase peptide synthesis (SPPS) was performed on a microwaveassisted synthesizer using standard Fmoc strategy in NMP on apolystyrene resin (TentaGel S Ram). HATU was used as coupling reagenttogether with DIPEA as base. Piperidine (20% in NMP) was used fordeprotection. Pseudoprolines: Fmoc-Phe-Thr(psiMe,Mepro)-OH andFmoc-Asp-Ser(psiMe,Mepro)-OH (purchased from NovaBiochem) were usedwhere applicable.

Abbreviations employed are as follows:

-   Boc: tert-butyloxycarbonyl-   ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methyl-butyl-   Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl-   DCM: dichloromethane-   DMF: N,N-dimethylformamide-   DIPEA: diisopropylethylamine-   EDT: 1,2-ethanedithiol-   EtOH: ethanol-   Et₂O: diethyl ether-   HATU:    N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminium    hexafluorophosphate N-oxide-   MeCN: acetonitrile-   NMP: N-methylpyrrolidone-   TFA: trifluoroacetic acid-   TIS: triisopropylsilane

Cleavage:

The crude peptide was cleaved from the resin by treatment with95/2.5/2.5% (v/v) TFA/TIS/water at room temperature (r.t.) for 2 hours.For peptides with a methionine in the sequence a mixture of 95/5 (v/v)TFA/EDT was used. Most of the TFA was removed at reduced pressure andthe crude peptide was precipitated and washed with diethylether andallowed to dry to constant weight at ambient temperature.

The following compounds were synthesised:

(Compound 1) H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLS-OH (Compound 2) H-HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-OH (Compound 3) H-HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLRA-OH (Compound 4)H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLLSA-NH₂(Compound 5) H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLESA-NH₂ (Compound 6) H-HSQGTFTSDYSRYLDS-K(Hexadecanoyl-isoGlu)-AAEDFVEWLLRA-NH₂ (Compound 7) H-HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAEDFVEWLLRA-NH₂ (Compound 8) H-HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLS-NH₂ (Compound 9) H-HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLR-OH (Compound 10) H-HSQGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-AAHEFVEWLESA-NH₂ (Compound 11)H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- RAKDFIEWLLS-OH(Compound 12) H-HSQGTFTSDYSRYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ (Compound 13)H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLSA-OH(Compound 14) H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-RAKDFIEWLLSA-OH (Compound 15)H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA-NH₂(Compound 16) H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAEDFVEWLESA-NH₂ (Compound 17)H-H-Aib-HGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEEFIEWLESA-OH(Compound 18) H-HSHGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAHEFIEWLESA-OH(Compound 19) H-H-Aib-HGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)-AAHEFVEWLESA-NH₂ (Compound 20)H-H-Aib-QGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA-NH₂(Compound 21) H-H-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)-AAEDFIEWLESA-NH₂ (Compound 22)H-HSQGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA-NH₂(Compound 23) H-H-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)-AAHDFVEWLESA-NH₂ (Compound 24)H-H-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEDFVEWLESA-NH₂(Compound 25) H-H-DSer-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-AAKDFIEWLESA-NH₂

Example 2: General Synthesis of Acylated Glucagon Analogues

The peptide backbone was synthesized as described above for the generalsynthesis of glucagon analogues, with the exception that it was acylatedon the side chain of a lysine residue with the peptide still attached tothe resin and fully protected on the side chain groups, except theepsilon-amine on the lysine to be acylated. The lysine to be acylatedwas incorporated with the use of Fmoc-Lys(ivDde)-OH or Fmoc-Lys(Dde)-OH.The N-terminus of the peptide was protected with a Boc group using Boc₂Oin NMP. While the peptide was still attached to the resin, the ivDdeprotecting group was selectively cleaved using 5% hydrazine hydrate inNMP. The unprotected lysine side chain was then first coupled with aspacer amino acid like Fmoc-Glu-OtBu, which was subsequently deprotectedwith piperidine and acylated with a fatty acid using standard peptidecoupling methodology as described above. Alternatively, the histidine atthe N-terminal may be incorporated from the beginning asBoc-His(Boc)-OH. Cleavage from the resin and purification were performedas described above.

Example 3: Glucagon Receptor and GLP-1-Receptor Efficacy Assays

The cDNA encoding either the human glucagon receptor (Glucagon-R)(primary accession number P47871) or the human glucagon-like peptide 1receptor (GLP-1R) (primary accession number P43220) were synthesized andcloned into a mammalian expression vector containing a Zeocin resistancemarker.

The mammalian expression vectors encoding the Glucagon-R or the GLP-1-Rwere transfected into Chinese hamster ovary (CHO) cells by theAttractene method method. Stably expressing clones were obtained byZeocin selection (250 μg/mL) upon limited dilution of cells resistant tothe selection pressure. Glucagon-R and GLP-1-R cell clones expressingwere picked, propagated and tested in the Glucagon-R and GLP-1-Refficacy assays as described below. One Glucagon-R expressing clone andone GLP-1-R expressing clone were chosen for compound profiling.

CHO cells expressing the human Glucagon-R, or human GLP-1-R were seeded24 hours prior to the assay at 30,000 cells per well in 96-wellmicrotiter plates in culture in 100 μl growth medium. On the day ofanalysis, growth medium was removed and the cells were washed once with200 μl of assay buffer (Krebs-Ringer-buffer—KRBH). The buffer wasremoved and the cells were incubated for 15 min at room temperature in10 μl KRBH (KRBH+10 mM HEPES, 5 mM NaHCO₃, 0.1% (V/V) BSA) with 0.1 mMIBMX in deionized water containing increasing concentrations of testpeptides. The reaction was stopped by the addition of lysis buffer (0.1%w/v BSA, 5 mM HEPES, 0.3% v/v Tween-20). After cell lysis for 10 min atroom temperature, lysates were transferred to 384-well plates and 10 μlof acceptor/donorbead mixture as contained in the AlphaScreen™ cAMPFunctional Assay Kit was added. After one hour of incubation at roomtemperature in the dark, the cAMP content was determined applying theAlphaScreen™ cAMP Functional Assay Kit from Perkin-Elmer according tomanufacturer instructions. EC₅₀ and relative efficacies compared toreference compounds (glucagon and GLP-1) were calculated applyingcomputer aided curve fitting. The GLP-1/glucagon ratio is calculated asdefined earlier. See Tables 1A and 1B.

TABLE 1A EC50 EC50 Ratio hGCGR hGLP-1R GLP-1/ Compound CHO-K1 [nM]CHO-K1 [nM] Glucagon 1 0.90 0.54 0.60 2 0.31 0.77 2.48 3 0.35 0.91 2.604 1.56 0.38 0.24 5 0.46 0.16 0.35 6 0.46 0.35 0.76 7 0.45 0.27 0.60 80.29 0.41 1.41 9 0.25 0.35 1.40 10 0.23 0.23 1.00 11 0.61 0.64 1.05 120.43 0.37 0.86 13 0.98 0.51 0.52 14 0.54 0.53 0.98 15 0.61 0.19 0.31 161.58 0.20 0.13

TABLE 1B EC50 EC50 Ratio hGCGR hGLP-1R GLP-1/ Compound CHO-K1 [nM]CHO-K1 [nM] Glucagon 17 0.05 nM 0.23 nM 4.6 18 0.14 nM 0.87 nM 6.2 190.08 nM 0.27 nM 3.38 20 0.68 nM 0.23 nM 0.34 21 1.32 nM 0.12 nM 0.09 220.10 nM 0.24 nM 2.40 23 0.25 nM 0.13 nM 0.52 24 0.92 nM 0.11 nM 0.12 250.16 nM 0.31 nM 1.94The two sets of compounds were tested in experiments performed using thesame protocol in different laboratories.

Example 4: Agonistic Activity on Endogenous GLP-1 Receptor

Agonistic activity of the test compounds on endogenous GLP-1 receptorswas determined using a murine insulinoma cell line. Intracellular cAMPwas used an indicator of receptor activation.

Cells were cultured for 24 h at a density of 10,000 cells/well in a384-well plate. Medium was removed and 10 μL KRBH buffer (NaCl 130 mM,KCl 3.6 mM, NaH₂PO₄ 0.5 mM, MgSO₄ 0.5 mM, CaCl₂) 1.5 mM) containing testcompound or GLP-1 (at increasing concentrations from 0.1 μM to 100 nM)or solvent control (0.1% (v/v) DMSO) was added to the wells for 15minutes at a temperature of 26° C.

The cellular cAMP content is measured using the AlphaScreen cAMPFunctional Assay Kit (Perkin Elmer). Measurement was performed using theEnvision (PerkinElmer) according to manufacturer's recommendations.

All measurements were performed in quadruplicate.

Results were converted into cAMP concentrations using a cAMP standardcurve prepared in KRBH buffer containing 0.1% (v/v) DMSO. The resultingcAMP curves were plotted as absolute cAMP concentrations (nM) over log(test compound concentration) and analyzed using the curve fittingprogram XLfit.

Parameters calculated to describe the both the potency as well as theagonistic activity of each test compound on the endogenous GLP-1receptors were:

pEC50 (negative logarithmic value of EC50, a concentration resulting ina half-maximal elevation of cAMP levels, reflecting the potency of thetest compound);

Percent control (% CTL)(% cAMP elevation for each test compoundconcentration normalized based on the GLP-1-induced maximum cAMPresponse (100% CTL)). See Table 2.

TABLE 2 EC50 GLP-1R (murine insulinoma Compound cells) [nM] 1 1.47 21.41 3 1.06 4 0.95 5 0.54 6 1.14 7 0.84 8 0.95 9 1.02 10 0.33 11 1.01 121.62 13 1.06 14 0.72 15 0.30 16 0.33 17 0.89 18 0.99 19 0.33 20 0.27 210.30 22 0.35 23 0.31 24 0.33 25 0.41

Example 5: Agonistic Activity on Endogenous Glucagon Receptor

Agonistic activity of the test compounds on endogenous glucagon receptorwas determined by measuring their effect on rate of glycogen synthesisin primary rat hepatocytes. Upon activation of the glucagon receptor, aninhibition of the glycogen synthesis rate is expected. Rate of glycogensynthesis was determined by counting the amount of radioactively labeledglucose incorporated into the cellular glycogen stores in a definedperiod of time.

Primary rat hepatocytes were cultured at a density of 40,000 cells/wellin a 24-well plate for 24 hours at 37° C. and 5% CO₂.

Medium was discarded and the cells washed with PBS. 180 μL of KRBH-basedbuffer containing 0.1% BSA and glucose at a concentration of 22.5 mM wasthen added to the wells, followed by test compound and 40 μCi/mlD-[U14C] glucose (20 μL each). Incubation was continued for 3 hours.

At the end of the incubation period, the incubation buffer was aspiratedand cells washed once with ice-cold PBS before lysis by incubation for30 min at room temperature with 100 μL 1 mol/l NaOH.

Cell lysates were transferred to 96-well filter plates and glycogenprecipitated by incubating the filter-plates for 120 min at 4° C.followed by washing the filter plates 4 times with ice-cold ethanol(70%). The resulting precipitates were filtered to dryness and theamount of incorporated ¹⁴C-glucose determined by using a Topcountscintillation counter according to manufacturer's recommendations.

Wells with vehicle controls (0.1% (v/v) DMSO in KRBH buffer) wereincluded as reference for non-inhibited glycogen synthesis (100% CTL).Wells without added D-[U¹⁴C] glucose were included as controls fornon-specific background signal (subtracted from all values). Endogenousglucagon peptide was used as a positive control.

All treatments were performed at least in triplicates.

Parameters calculated to describe the both the potency as well as theagonistic activity of each test compound on the endogenous glucagonreceptor are pEC50 and % CTL. % CTL is determined by calculating thepercentage of CPM/well in the presence of the test compound compared tothe CPM/well of the vehicle control after subtracting the backgroundCPM/well:[CPM/well(basal)−CPM/well(sample)]*100/[CPM/well(basal)−CPM/well(control)]

An activator of the glucagon receptor will result in an inhibition ofthe glycogen synthesis rate and will give % CTL values between 0% CTL(complete inhibition) and 100% CTL (no observable inhibition).

The resulting activity curves were plotted as absolute counts (unit:cpm/sample) over log (test compound concentration) and analyzed usingthe curve fitting program XLfit.

pEC50 (negative logarithmic value of EC50) reflects the potency of thetest compound.

TABLE 3 EC50 GLP-1R Compound Rat hepat. [nM] 1 0.13 1 0.02 2 0.43 3 0.164 0.35 5 0.03 6 2.87 7 1.92 8 1.10 9 0.37 10 0.04 12 0.24 13 0.67 140.10 15 0.17 16 1.85 17 0.18 18 0.18 19 0.06 20 0.39 21 3.40 22 0.28 231.32 24 6.15 25 0.15

The terms EC₅₀ and pEC₅₀ quoted in relation to GLP-1R activation couldequally be regarded as IC₅₀ and pIC₅₀ in relation to glycogen synthesis.

Example 6: Estimate of Pharmacokinetic Parameters

Pharmacokinetic parameters of the test compounds were determined aftersubcutaneous and intravenous administration to C57Bl/6J mice.

8 week old male C57Bl/6J mice were obtained from Taconic (Denmark)weighing approximately 20-25 g at time of arrival at the test facility.The mice were caged in European standard cages type III with light cycleof 12-hour dark and 12-hour light (lights on 06.00). Both diet, Altromin1324 (Altromin, Germany), and water was administered ad libitum duringthe whole experimental period. The 8-11 week animals were imported tothe laboratory at least 7 days before the start up of the experimentalprocedure in order to assure proper acclimatization. After sampling themice were euthanized by cervical dislocation.

The compounds were first dissolved in 0.096% aqueous ammonia to anominal concentration of 2 mg/ml, and then diluted to the desired dosingstrength (4 μM) in sterile PBS containing 25 mM phosphate buffer, pH7.4. Subcutaneous and intravenous injections corresponding to 20 nmol/kgwere given. The GLP-1-glucagon dual agonists were administered forsubcutaneous dosing in the neck region and via a lateral tail vein forintravenous dosing.

Blood samples (250 μl) were collected from the periorbital plexus attime points 0.5, 2, 4, 6, 12, 16, 20, 24 h for subcutaneous and 10, 30min, 3, 6, 12, 18, 24 h for intravenous administration into ice-chilledK₃EDTA tubes and centrifuged for 5 minutes at 4° C. within 20 minutes ofsampling. Plasma (>100 μl) was separated to ice-chilled Micronic tubes,immediately frozen, and kept at −70° C. until analysed for plasmaconcentration for the respective GLP-1-glucagon compound using LC-MS/MS.Individual plasma concentration-time profiles were analysed by anon-compartmental approach in WinNonLin v6.3 (Pharsight inc, MountainView, Calif., USA), and the resulting pharmacokinetic parametersdetermined. See Table 4.

TABLE 4 SC IV Mean Bio- Terminal Terminal Com- Residence availabilityTmax half Clearance half life pound Time (hr) (%) (hr) life (hr)(L/hr/kg) (hr) 1 12.6 52 4 6.2 0.0102 5.2 2 11.2 59 4 5.1 0.0111 5.0 3 9.8 52 6 3.9 0.0115 4.1 4 12.5 53 4 6.4 0.0111 7.4 5  7.6 81 4 3.80.0111 4.0 6 10.6 47 4 4.3 0.0094 3.7 7 12.8 75 4 8.6 0.0132 5.5 8  8.142 4 4.1 0.0077 3.9 9  4.7 34 2 2.9 0.0383 0.9 10   6.5 80 2 3.7 0.02133.0 11  12.5 64 4 5.0 0.0094 3.9 12   8.3 43 6 4.3 0.0135 4.1 13  14.776 4 8.6 0.0089 6.3 14  12.2 64 4 6.1 0.0135 6.2 15  17.4 97 6 10.5 0.0077 6.0 16   7.5 80 2 5.0 0.0187 4.8

Example 7: Oral Glucose Tolerance Test (OGTT) in C57BL/6J Mice

Male C57BL/6J mice were fasted for 10 h before they received an oralglucose bolus of 2 g/kg body weight. Peptides were dissolved in salinebuffered with 25 mM phosphate and administered at a dose of 10 nmol/kgbody weight by subcutaneous injection 4 h before the glucose bolus.Control animals received a vehicle injection only. Group size was 7animals per group. A pre-dose and a pre-glucose (0 min) blood samplewere obtained by tail bleeding and blood glucose was measured with aglucometer. Additional samples of tail blood for measuring blood glucosewere obtained 15 min, 30 min, 60 min, 90 min and 120 min after theglucose challenge. Glucose excursion was quantified by calculating thetotal area under the blood glucose-time curve (AUC) between 0 min and120 min. Calculation of AUC was done by the trapezoidal rule withoutbaseline correction. The data are presented as mean±S.E.M. Statisticalcomparisons were conducted by one-way ANOVA followed by Tukey's posttest. Vehicle alone was used as a control. See Table 5.

TABLE 5 Compound AUC (% Ctrl) 1 66 2 66 3 70 4 65 5 46 6 50 7 57 8 50 957 10 46 11 61 12 56 13 55 14 59 15 42 16 44

Example 8: Sub-Chronic Effects of Glucagon-GLP-1 Receptor Dual ActingAgonists on Adiposity in Diet-Induced Obese C57BL/6J Mice

Male C57BL/6J mice (Taconic A/S, Denmark) at an age of 5 weeks weremaintained on a 12:12 hour light-dark cycle on a high-fat diet (60% oftotal energy from fat, D12492, Research Diet Inc.) for 20 weeks. Allmice (housed 2 per cage) were then mock-treated for a week toacclimatize the animals to handling and injections. Subsequently, themice were stratified according to body fat mass (measured by magneticresonance technique) and body weight into 9 groups (n=9-12). Animalswere thereafter treated twice daily with subcutaneous injections (5ml/kg) of vehicle (25 mM phosphate buffer, pH 7.4) or test substances (5nmol/kg per administration, in equivalent amount of vehicle) for a totalof 31 days. The daily injections were given at the start and at the endof the light phase. Body weight, food and water intake were determineddaily throughout the study. The data are presented in Table 6 as meanbody weight change±S.E.M. S.E.M. is defined as standard error of themean and was calculated using the formula: S.E.M.=SD/square root (n),where SD is the standard deviation and n the number of observations (inthis case animals/group).

Statistical analyses were performed using Graph Pad Prism version 5. Themeasured parameters were compared using one-way ANOVA followed byDunnett's multiple comparison test. Differences were consideredstatistically significant at p<0.05.

TABLE 6 Mean body Significance of Treatment weight difference vs. Groupchange (%) SEM vehicle group Vehicle 4.8 1.3 Liraglutide −2.3 1.5 **Compound 2 −5.1 1.9 *** Compound 7 −8.7 1.5 *** Compound 14 −9.5 1.1 ***Compound 15 −10.9 1.6 *** Compound 16 −12.2 1.1 *** ** - p <0.01, *** -p <0.001 vs vehicle

The invention claimed is:
 1. A compound having the formula:R¹—X—OH wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl ortrifluoroacetyl; and X is a peptide which has the formula:(SEQ ID NO: 3) HSQGTFTSDYSKYLDSKAAHDFVEWLLRA (SEQ ID NO: 13)H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLSA (SEQ ID NO: 14)H-Aib-QGTFTSDYSKYLDEKRAKDFIEWLLSA

or a pharmaceutically acceptable salt thereof.
 2. A compound having theformula:R¹—X—Z—R² wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl ortrifluoroacetyl; R² is OH or NH₂; X is a peptide of SEQ ID NO: 15:(SEQ ID NO: 15) H-Aib-QGTFTSDYSKYLDEKAAKDFIEWLESA

or which differs from SEQ ID NO: 15 at up to three of the followingpositions whereby, if different from SEQ ID NO: 15: the residue atposition 2 is selected from Ser and D-Ser the residue at position 3 isselected from His and Hse the residue at position 12 is Arg the residueat position 15 is Glu the residue at position 16 is Ser the residue atposition 18 is Arg the residue at position 20 is selected from His andGlu the residue at position 21 is Glu the residue at position 23 is Valthe residue at position 28 is selected from Arg and Glu the residue atposition 29 is absent; and Z is absent or is a sequence of 1-20 aminoacid units independently selected from the group consisting of Ala, Leu,Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn; or apharmaceutically acceptable salt thereof.
 3. A compound according toclaim 2 wherein the residue at position 20 is His.
 4. A compoundaccording to claim 2 the formula:R¹—X—Z—R² wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl ortrifluoroacetyl; R² is OH or NH₂; X is a peptide of SEQ ID NO: 15:(SEQ ID NO: 15) H-Aib-QGTFTSDYSKYLDEKAAKDFIEWLESA

or which differs from SEQ ID NO: 15 at one or both of the followingpositions whereby, if different from SEQ ID NO: 15: the residue atposition 21 is Glu the residue at position 23 is Val and Z is absent oris a sequence of 1-20 amino acid units independently selected from thegroup consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu,Dpr and Orn; or a pharmaceutically acceptable salt thereof.
 5. Acompound having the formula:R¹—X—Z—R² wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl ortrifluoroacetyl; R² is OH or NH₂; X is a peptide of SEQ ID NO: 16:(SEQ ID NO: 16) H-Aib-QGTFTSDYSKYLDSKAAEDFVEWLESA

or which differs from SEQ ID NO: 16 at up to three of the followingpositions whereby, if different from SEQ ID NO: 16: the residue atposition 2 is selected from Ser and D-Ser the residue at position 3 isselected from His and Hse the residue at position 12 is Arg the residueat position 15 is Glu the residue at position 16 is Glu the residue atposition 18 is Arg the residue at position 20 is selected from Lys andHis the residue at position 21 is Glu the residue at position 23 is Ilethe residue at position 28 is selected from Arg and Glu the residue atposition 29 is absent and Z is absent or is a sequence of 1-20 aminoacid units independently selected from the group consisting of Ala, Leu,Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn; or apharmaceutically acceptable salt thereof.
 6. A compound according toclaim 5 the formula:R¹—X—Z—R² wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl ortrifluoroacetyl; R² is OH or NH₂; X is a peptide of SEQ ID NO: 16:(SEQ ID NO: 16) H-Aib-QGTFTSDYSKYLDSKAAEDFVEWLESA

or which differs from SEQ ID NO: 16 at one or both of the followingpositions whereby, if different from SEQ ID NO: 16: the residue atposition 21 is Glu the residue at position 23 is Ile and Z is absent oris a sequence of 1-20 amino acid units independently selected from thegroup consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu,Dpr and Orn; or a pharmaceutically acceptable salt thereof.
 7. Acompound according to claim 1 wherein one or more of the amino acid sidechains in the peptide X or Z is conjugated to a lipophilic substituent.8. A compound according to claim 7 wherein peptide X has the formula:HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLRA;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLSA; orH-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- RAKDFIEWLLSA.


9. A compound according to claim 8 having the formula: (Compound 3)H-HSQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLRA-OH (Compound 13)H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLSA-OH or(Compound 14) H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-RAKDFIEWLLSA-OH

or a pharmaceutically acceptable salt thereof.
 10. A compound accordingclaim 2 wherein one or more of the amino acid side chains in the peptideX or Z is conjugated to a lipophilic substituent.
 11. A compoundaccording to claim 10 wherein peptide X has the formula:H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESAH-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAEDFVEWLESAH-Aib-QGTFTSDYSKYLEEK-(Hexadecanoyl-isoGlu)- AAKDFIEWLESAH-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEDFIEWLESAHSQGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA orH-DSer-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA.


12. A compound according to claim 11 the formula: (Compound 15)H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA-NH₂(Compound 16) H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAEDFVEWLESA-NH₂ (Compound 20)H-H-Aib-QGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA-NH₂(Compound 21) H-H-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)-AAEDFIEWLESA-NH₂ (Compound 22)H-HSQGTFTSDYSKYLEE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA-NH₂(Compound 25) H-H-DSer-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-AAKDFIEWLESA-NH₂

or a pharmaceutically acceptable salt thereof.
 13. A compound accordingclaim 5 wherein one or more of the amino acid side chains in the peptideX or Z is conjugated to a lipophilic substituent.
 14. A compoundaccording to claim 13 wherein peptide X has the formula:H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESAH-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAEDFVEWLESAH-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEDFIEWLESAH-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAHDFVEWLESA orH-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEDFVEWLESA.


15. A compound according to claim 14 having the formula: (Compound 15)H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAKDFIEWLESA-NH₂(Compound 16) H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAEDFVEWLESA-NH₂ (Compound 21)H-H-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEDFIEWLESA-NH₂(Compound 23) H-H-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)-AAHDFVEWLESA-NH₂ (Compound 24)H-H-Aib-QGTFTSDYSKYLES-K(Hexadecanoyl-isoGlu)- AAEDFVEWLESA-NH₂

or a pharmaceutically acceptable salt thereof.